Case VI.1, from family C, was a female of apparently healthy second cousin parents. She was diagnosed with unclassified cardiomyopathy at the age of 2.5 years and died at the age of 8 years of SCD. Family history was positive for cardiac disease and occurrence of SCD in three family members (V.5, V.6, and V.8), who had been diagnosed with unclassified cardiomyopathy between the ages of 2 and 3 and had died between 5 and 9 years of age (Fig. 1c). There was no record of cardiac evaluation available for these individuals. The 40-year-old mother (V.2) was offered genetic testing following the death of her daughter (VI.1). Unfortunately, there was no tissue available from the deceased individual VI.1.
Case#: Family C, Case VI.1; female; diagnosed at age 2.5 years; died at age 8
DiseaseAssertion: Unclassified cardiomyopathy
FamilyInfo: Family C has a positive family history of childhood-onset cardiomyopathy and sudden cardiac death. The affected individual (VI.1) was diagnosed with cardiomyopathy at age 2.5 years and died at age 8. Three maternal relatives (V.5, V.6, and V.8) are depicted as affected and deceased in the pedigree (Fig. 1C). Both parents (V.1 and V.2) were clinically unaffected heterozygous carriers of the MYL3 c.482-1G>A splice-acceptor variant. The genotype of VI.1 could not be confirmed because DNA was unavailable.
ParentalTesting: PCR and bidirectional Sanger sequencing confirmed that both parents (V.1 and V.2) were heterozygous carriers of MYL3 c.482-1G>A. Direct testing of the affected daughter (VI.1) was not possible because DNA was unavailable. Her homozygous genotype was therefore suspected but not confirmed (Fig. 1C; Supplementary Genetic Findings).
CasePresentingHPOs: HP:0001638 (Cardiomyopathy); HP:0001645 (Sudden cardiac death).
CaseHPOFreeText: Female diagnosed with unclassified cardiomyopathy at age 2.5 years; died of sudden cardiac death at age 8 (Family C, Fig. 1C).
CaseNotHPOs: NR (no patient-specific negative HPO findings confirmed for VI.1).
CaseNotHPOFreeText: The authors reported no evidence of muscle weakness by neurological examination or history among affected individuals from the three families; however, it is unclear whether this assessment specifically included VI.1.
CasePreviousTesting: NR (no prior genetic testing reported for VI.1).
Genotyping Method: Genetic analysis of the affected individual's mother (V.2) identified MYL3 c.482-1G>A. PCR and bidirectional Sanger sequencing validated the variant and confirmed both parents (V.1 and V.2) as heterozygous carriers. VI.1 was not directly genotyped because DNA was unavailable (Supplementary Genetic Findings; Fig. 1C and S2C).
FunctionalAnalysis: A minigene splicing assay in human HEK293FT cells demonstrated that MYL3 c.482-1G>A causes abnormal RNA splicing. The predominant outcome (~75%) was skipping of the 78-bp exon 5, producing an in-frame deletion (p.Gly161_Glu186del). A less frequent outcome (~25%) was deletion of the first nucleotide of exon 5, causing a frameshift (p.Gly161Valfs*4). Both outcomes are predicted to disrupt EF-hand domains. The authors note that the minigene assay may not fully reflect in vivo splicing frequencies (Supplementary Results, pp. 7–8; Fig. 2E–I).
Variant: MYL3 c.482-1G>A (rs727503300)
ClinVar: 164486
CAID: CA013932
gnomAD: NR MYL3 c.482-1G>A was not found in the gnomAD v4 (GRCh38) region results at chr3:46,860,813. Allele frequency not reported for this variant in the searched dataset.
AdditionalInfo: The affected individual (VI.1) died at age 8, and DNA was unavailable for direct genetic testing. Both clinically unaffected parents (V.1 and V.2) were confirmed heterozygous carriers of MYL3 c.482-1G>A, suggesting that VI.1 may have inherited the variant from both parents; however, her genotype remains unconfirmed. A minigene assay demonstrated that the variant disrupts RNA splicing, predominantly causing exon 5 skipping, with a less frequent frameshift-producing splicing event. These functional findings support a damaging effect of the variant but do not establish VI.1's genotype (Fig. 1C; Fig. 2E–I; Supplementary Genetic Findings and Minigene Assay Results).